This invention relates to image analysis, and, more particularly, ray-based image analysis for biological specimens.
The process of evaluating the effect of various compounds on biological specimens for drug screening and other research activities is generally labor intensive. In even a relatively modest cell-based assay, one may investigate hundreds or thousands of different combinations of compounds and concentrations. One approach for gathering and analyzing such large amounts of data involves using highly automated equipment to expose cell-based biological specimens to different conditions. Automated microscopy equipment and digital image processing software may be used to acquire digital images of each of the specimens and to evaluate the impact of the different conditions to which the specimens have been exposed.
It is an object of the present invention to provide improved systems and methods for evaluating images of biological specimens.
It is another object of the present invention to provide improved image processing arrangements that may be used in performing cell-based assays.
These and other objects of the invention are accomplished in accordance with the principles of the present invention by providing image processing equipment and methods suitable for evaluating images of biological specimens. A user of the system may perform cell-based assays for drug screening or general biological research. Cells may be cultured in a number of individual wells in microtiter plates or other suitable biological specimen mounting arrangements may be used. The cells in each well may be individually tested. For example, the cells in each well may be exposed to different biochemical agents such as peptides, enzymes, nucleic acids, or other suitable organic or inorganic compounds.
Digital images of the cells may be collected using automated equipment. The automated equipment may, for example, acquire an image corresponding to the cells in each microtiter well. If desired, the cells may be marked with fluorescent markers and images may be acquired using a single-wavelength or multi-wavelength scanning laser microscope and a digital image sensor. The images may also be acquired with other suitable microscopes (e.g., light microscopes) or other image acquisition equipment.
The digital images of the cells may be processed to determine the location of cell nuclei. A seed point within each nucleus may be identified. A set of radially-oriented rays may be associated with each cell nuclei. The rays associated with a given cell may have a common origin that is located at the seed point. If desired, the rays may each have a different origin. As an example, each ray may originate from a different location on the boundary of the nucleus. Each ray may extend radially outward from the cell interior towards the outer cell boundary until terminated at an outer endpoint.
The rays preferably do not extend beyond a predefined maximum extent and do not overlap with the nuclei of cells other than the cell from which the rays originate. A user of the system may adjust image acquisition and analysis parameters such as the ray termination criteria that specify how the system is to determine the outer endpoints of each ray. Suitable user-specified termination criteria include threshold-based criteria and maximum length criteria.
Once the locations of the rays corresponding to each seed point in an image have been determined, various ray-based image analysis steps may be performed using the image data that lies along the rays. For example, average pixel intensity levels may be determined along the rays or portions of the rays. Minimum and maximum pixel intensity levels, statistical intensity level distributions, and ratios of any of such values may also be determined.
When a multi-wavelength fluorescence microscope arrangement is used, images may be acquired using different data channels each of which corresponds to a different fluorescent marker color. For example, one data channel may be used to capture image data for a blue wavelength. Another data channel may be used to capture red data and yet another data channel may be used to capture green data. With this type of approach, the DNA in the cell nuclei may be marked with a blue marker (for example) that binds to DNA. The blue channel data may be processed to identify nuclei and seed point locations. A green marker (for example) may be used to label a protein of interest. When the cell is exposed to a test compound, the green-labeled protein may or may not translocate within the cell. As an example, the green-labeled protein might initially be located on the outer cell wall. After being exposed to a particular compound, the green-labeled protein may be redistributed throughout the cytoplasm of the cell. This movement of the green-labeled protein may be analyzed using the ray-based analysis approach.